Imaging Breast Microcalcifications Using Dark-Field Signal in Propagation-Based Phase-Contrast Tomography.

Breast microcalcifications are an important primary radiological indicator of breast cancer. However, microcalcification classification and diagnosis may be still challenging for radiologists due to limitations of the standard 2D mammography technique, including spatial and contrast resolution. In this study, we propose an approach to improve the detection of microcalcifications in propagation-based phase-contrast X-ray computed tomography of breast tissues. Five fresh mastectomies containing microcalcifications were scanned at different X-ray energies and radiation doses using synchrotron radiation. Both bright-field (i.e. conventional phase-retrieved images) and dark-field images were extracted from the same data sets using different image processing methods. A quantitative analysis was performed in terms of visibility and contrast-to-noise ratio of microcalcifications. The results show that while the signal-to-noise and the contrast-to-noise ratios are lower, the visibility of the microcalcifications is more than two times higher in the dark-field images compared to the bright-field images. Dark-field images have also provided more accurate information about the size and shape of the microcalcifications.

Innate immune cells and bacterial infection in zebrafish.

The physical attributes of the zebrafish, including optical transparency during embryogenesis, large clutch sizes, external development, and rapid organogenesis were features that initially attracted developmental biologists to use this vertebrate as an experimental model system. With the progressive development of an extensive genetic "tool kit" and an ever-growing number of transgenic reporter lines, the zebrafish model has evolved into an informative system in which to mimic and study aspects of human disease, including those associated with bacterial infections. This chapter provides detailed protocols for microinjection of bacterial strains into zebrafish larvae and subsequent experiments to investigate single-larva bacterial burdens, live imaging of specific neutrophil and macrophage bactericidal functions, and how these protocols may be applied to drug discovery approaches to uncover novel immunomodulatory drugs.

Absolute x-ray dosimetry on a synchrotron medical beam line with a graphite calorimeter.

PURPOSE: The absolute dose rate of the Imaging and Medical Beamline (IMBL) on the Australian Synchrotron was measured with a graphite calorimeter. The calorimetry results were compared to measurements from the existing free-air chamber, to provide a robust determination of the absolute dose in the synchrotron beam and provide confidence in the first implementation of a graphite calorimeter on a synchrotron medical beam line. METHODS: The graphite calorimeter has a core which rises in temperature when irradiated by the beam. A collimated x-ray beam from the synchrotron with well-defined edges was used to partially irradiate the core. Two filtration sets were used, one corresponding to an average beam energy of about 80 keV, with dose rate about 50 Gy/s, and the second filtration set corresponding to average beam energy of 90 keV, with dose rate about 20 Gy/s. The temperature rise from this beam was measured by a calibrated thermistor embedded in the core which was then converted to absorbed dose to graphite by multiplying the rise in temperature by the specific heat capacity for graphite and the ratio of cross-sectional areas of the core and beam. Conversion of the measured absorbed dose to graphite to absorbed dose to water was achieved using Monte Carlo calculations with the EGSnrc code. The air kerma measurements from the free-air chamber were converted to absorbed dose to water using the AAPM TG-61 protocol. RESULTS: Absolute measurements of the IMBL dose rate were made using the graphite calorimeter and compared to measurements with the free-air chamber. The measurements were at three different depths in graphite and two different filtrations. The calorimetry measurements at depths in graphite show agreement within 1% with free-air chamber measurements, when converted to absorbed dose to water. The calorimetry at the surface and free-air chamber results show agreement of order 3% when converted to absorbed dose to water. The combined standard uncertainty is 3.9%. CONCLUSIONS: The good agreement of the graphite calorimeter and free-air chamber results indicates that both devices are performing as expected. Further investigations at higher dose rates than 50 Gy/s are planned. At higher dose rates, recombination effects for the free-air chamber are much higher and expected to lead to much larger uncertainties. Since the graphite calorimeter does not have problems associated with dose rate, it is an appropriate primary standard detector for the synchrotron IMBL x rays and is the more accurate dosimeter for the higher dose rates expected in radiotherapy applications.

Synchrotron-based in vivo tracking of implanted mammalian cells.

We have developed an X-ray imaging protocol that permits 3D visualisation of a small number of implanted cells within bulk tissue. The cells are marked using natural endocytosis of inert gold nano-particles. The resulting local increase in electron density allows high imaging contrast to be obtained from small clusters of these marked cells. Using this technique we have imaged C6 glioma cells within the brain of a model animal. The cells were marked by exposing them to colloidal gold incorporated in the growth media. Gold-loaded glioma cells were implanted into the brains of adult male Wistar rats. After tumours had been allowed to develop for up to 2 weeks, the animals were sacrificed and images of the intact cranium were acquired at the SYRMEP imaging station on the Elettra synchrotron in Italy. Computed tomography was performed using mixed absorption and phase contrast techniques at an X-ray energy of 24 keV. In the resulting volume datasets the tumour bulk is clearly visible and the infiltrating nature of the malignant growth is well demonstrated. Although the protocol was developed using this particular model of malignant brain tumour, it is believed that it will be possible to use it with other cell lines.

Phase contrast X-ray imaging of mice and rabbit lungs: a comparative study.

The significant degree of X-ray phase contrast created by air-tissue interfaces, coupled with the poor radiographic contrast of conventional chest radiographs, makes the inflated lung an ideal candidate for investigating the potential diagnostic improvement afforded by phase contrast X-ray imaging. In small animals these methods highlight the lung airways and lobe boundaries and reveal the lung tissue as a speckled intensity pattern not seen in other soft tissues. We have compared analyser-based and propagation-based phase contrast imaging modalities, together with conventional radiographic imaging, to ascertain which technique shows the greatest image enhancement for various lung sizes. The conventional radiographic image of a mouse was obtained on a Siemens Nova 3000 mammography system, whilst phase contrast images of mice and rabbit chests were acquired at the medical imaging beamline (20B2) at the SPring-8 synchrotron radiation research facility in Japan. For mice aged 1 day, 1 week and 1 month old it was determined that analyser-based imaging showed the greatest overall image contrast, however, for an adult rabbit both techniques yielded excellent contrast. The success of these methods in creating high quality images for rabbit lungs raises the possibility of improving human lung imaging using phase contrast techniques.

A preliminary study of breast cancer diagnosis using laboratory based small angle x-ray scattering.

Breast tissue collected from tumour samples and normal tissue from bi-lateral mastectomy procedures were examined using small angle x-ray scattering. Previous work has indicated that breast tissue disease diagnosis could be performed using small angle x-ray scattering (SAXS) from a synchrotron radiation source. The technique would be more useful to health services if it could be made to work using a conventional x-ray source. Consistent and reliable differences in x-ray scatter distributions were observed between samples from normal and tumour tissue samples using the laboratory based 'SAXSess' system. Albeit from a small number of samples, a sensitivity of 100% was obtained. This result encourages us to pursue the implementation of SAXS as a laboratory based diagnosis technique.

Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows.

High protein diets, which lead to excess production of nonprotein nitrogen such as ammonia and urea, have been associated with reduced fertility in dairy cows. In this study we test the hypothesis that diets containing high levels of quickly degradable urea nitrogen (QDN) compromise embryo development. Lactating dairy cows were fed mixed silage and concentrates twice daily. At 60 days postpartum, a synchronized estrus was induced and the cows were subsequently superovulated and inseminated using a standard protocol. On Day 7 after insemination, the uteri were flushed and embryos retrieved. At the start of treatment, cows were randomly allocated into three nutritional groups: control (CONT, n = 8), long (L-) QDN (n = 8) and short (S-) QDN (n = 9). The L-QDN cows were fed a supplement of urea from 10 days before insemination, and the S-QDN cows were fed the supplement from insemination until embryo collection. Both L- and S-QDN diets produced significant increases in plasma ammonia and urea 3 h post-feeding. The S-QDN but not the L-QDN diet was associated with a significant reduction in embryo yield. Embryo quality was also significantly reduced in the S-QDN cows. This study indicates that there is no deleterious effect on the yield and quality of embryos recovered 7 days after breeding when QDN feeding is initiated during the previous midluteal phase. However, introduction of a similar diet 10 days later, at the time of insemination, was deleterious. We suggest that QDN is toxic to embryos but cows can adjust within 10 days.

Breast cancer diagnosis using scattered X-rays.

Small-angle X-ray diffraction data has been collected from 99 ;core-cut' breast tissue specimens representing a number of different pathologies. Data in the range 75-1390 A have been compared with controls from patients with no breast disease. Bessel functions and Bragg maxima resulting from the fibrillar structure of collagen have been identified. The Bragg maxima indexed onto a 649 A lattice. Systematic differences in the intensities and D-spacings between the collagen of malignant, benign and normal tissue groups have been clearly demonstrated and quantified. These differences appear to be due to a significantly lower structural order within the malignant tissues. Possible explanations for this are discussed and the potential for utilizing this observation in cancer diagnosis is considered.

Comparison of three non-culture techniques for detection of Chlamydia trachomatis in genital tract specimens.

A direct immunofluorescence (DIF) technique (Imagen) and two enzyme immunoassay (EIA) techniques (Chlamydiazyme and IDEIA) were compared for the detection of Chlamydia trachomatis in genital specimens from 502 attenders at a genitourinary medicine clinic. Eighty-two attenders were regarded as infected: 67 with positive results by at least two of the three techniques and 15 by virtue of elementary bodies detected in stored EIA buffer samples. With a positivity criterion of greater than or equal to 6 bodies Imagen was 76% sensitive for men and 61% sensitive for women. The sensitivity of Chlamydiazyme was 73% for men and 90% for women; comparative values for IDEIA were 80% and 71%, respectively. All three techniques were over 98% specific. Sampling order appeared to influence the sensitivity of IDEIA for specimens from men. All three techniques were less sensitive in the absence of cervicitis. The performances of the EIA techniques compared favourably with that of the more established technique of DIF.

Effect of gonadal hormones on the incorporation of [3H]lysine into proteins of human fetal brain.

Samples were obtained from the hypothalamus, temporal lobe and frontal cortex of brains from human fetuses aged 14-22 weeks. Slices were prepared and incubated with estradiol, testosterone or dihydrotestosterone, and the incorporation of [3H]lysine into protein was compared with that of untreated control tissue. Cytosol and crude nuclear subcellular fractions from each area were made and resolution of specific proteins was carried out using a polyacrylamide gel system. Steroid hormones had no detectable effect on total protein synthesis, but incorporation of [3H]lysine into certain low molecular weight proteins of the hypothalamus increased significantly in the presence of estradiol and testosterone. Dihydrotestosterone was inactive. The temporal lobe and frontal cortex were unaffected by the steroid hormones.